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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to a class of monoclonal antibody that specifically binds to alpha-synuclein for use in combinational therapies, as well as to methods of using these in the treatment of synucleinopathies.
• Synucleinopathies also known as Lewy body diseases (LBDs) are characterized by deposition of intracellular protein aggregates that are microscopically visible as Lewy bodies (LBs) and/or Lewy neurites, where the protein alpha-synuclein is the major component (Jellinger, Mov Disord. 2012 Jan;27(1 ):8-30; McKeith et al., Neurology (1996) 47:1 1 13-24).
• Synucleinopathies include Parkinson's disease (PD) (including idiopathic and inherited forms of Parkinson's disease) and Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease (CAPD), pure autonomic failure (PAF) and multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy-Drager Syndrome)).
• PD Parkinson's disease
• DLB Diffuse Lewy Body
• LBV Dementia with Lewy Bodies
• LBV Lewy body variant of Alzheimer's disease
• CAPD Combined Alzheimer's and Parkinson disease
• MSA multiple system atrophy
• Olivopontocerebellar Atrophy Striatonigral Degeneration and Shy-Drager Syndrome
• Synucleinopathies frequently have degeneration of the dopaminergic nigrostriatal system, responsible for the core motor deficits in Parkinsonism (rigidity, bradykinesia, resting tremor), but there is also widespread occurrence of Lewy bodies and dystrophic Lewy neurites in the central, peripheral and autonomic nervous system and brain regions and other organs associated with non-motor dysfunctions, such as dementia and autonomic nervous system deficits.
• Several of the non-motor signs and symptoms are thought to precede motor symptoms in Parkinson's disease and other synucleinopathies. Such early signs include, for example, REM sleep behaviour disorder (RBD) and loss of smell and constipation (Mahowald et al., Neurology (2010) 75:488-489).
• Synucleinopathies continue to be a common cause for movement disorders and cognitive deterioration in the aging population (Galasko et al., Arch. Neurol. (1994) 51 :888-95).
• Alpha-synuclein is a member of a family of proteins including beta- and gamma-synuclein and synoretin. Alpha-synuclein is expressed in the normal state associated with synapses and is believed to play a role in regulating synaptic vesicle release and thereby affecting neural communication, plasticity, learning and memory. [0005] Several studies have implicated alpha-synuclein with a central role in PD pathogenesis. The protein can aggregate to form intracellular insoluble fibrils in pathological conditions.
• synuclein accumulates in LBs (Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synuclein gene as well as duplications and triplications of the gene co-segregate with rare familial forms of parkinsonism (Kruger et al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-7).
• alpha-synuclein can be secreted into the extracellular fluid and be present in plasma and cerebrospinal fluid (CSF).
• CSF cerebrospinal fluid
• mice Over expression of alpha-synuclein in transgenic mice mimics some pathological aspects of Lewy body disease.
• Several different transgenic lines of mice over-expressing alpha-synuclein have been generated in the last ten years (described in reviews: Koehler et al 2014, PLoS One. 2013 May 31 ;8(5):e64649; Fleming and Chesselet, 2006,Behav Pharmacol. 2006 Sep;17(5-6):383-91 ; Springer and Kahle 2006,Curr Neurol Neurosci Rep. 2006 Sep;6(5):432-6).
• alpha-synuclein gene and posttranslational modifications of the protein can create alpha-synuclein protein forms that have enhanced capacity to form aggregated and/or toxic forms of alpha-synuclein (Beyer and Ariza, Mol Neurobiol. 2013 Apr;47(2):509-24).
• the precise pathological species of alpha-synuclein remains unknown.
• Various misfolded/aggregated/secreted species ranging from oligomers to fibrils, and different post-translational modifications have been associated with toxicity but there is no consensus on which is most important, if indeed there even is a single toxic species.
• a C- terminal antibody 274 which targets an epitope that involves alpha-synuclein residues 120-140 was also shown to have an effect in a preclinical model on spreading of the pathology from cell to cell.
• antibodies targeting conformational species such as oligomers and fibrils of alpha-synuclein have been shown to be able to at least reduce the levels of these presumably toxic alpha-synuclein species (Lindstrom et al. 2014, Neurobiol Dis. 2014 Sep;69:134-43 and Spencer et al. 2014, Mol Ther. 2014 Oct;22(10): 1753-67).
• a therapeutic antibody should be ideally able to bind to most of the alpha-synuclein species that are formed by alternative splicing or posttranslational modifications, such as truncations, as well as oligomeric and fibrillary forms.
• amino acids that are important for binding of 9E4 are asparagine 122 and tyrosine 125 (according to an alanine scan presented in patent US20140127131 ), and this means that this antibody cannot bind alpha-synuclein which is truncated at amino acids 1 19, and 122, which are some of the major truncated species in Parkinson brain tissue (Kellie et al. Sci Rep. 2014;4:5797). The same would be the case for the antibody 274 and antibody mab47 (US8,632,776).
• amino terminal antibodies would possibly not be able to bind to some of the major truncated species that lack the first amino acids of alpha-synuclein, such as alpha-synuclein truncated to amino acids 5-140.
• one suggested mechanism of action is the prevention of truncation at amino acids 1 19-122 in extracellular space, as the antibody will bind to the same region where the protease that will cleave alpha- synuclein (Games et al. 2014, J Neurosci. 2014 Jul 9;34(28):9441 -54).
• a similar mechanism of action could also be found with antibodies in close proximity of the site, and therefore many antibodies around this region would be expected to have this activity.
• calcium/calmodulin-dependent protein kinase II alpha (CamKII-alpha) promoter was associated with alpha-synuclein aggregation and a progressive deficit in cortical- hippocampal memory tests including the Barnes maze and novel object recognition (Hall et al. 2015, Exp Neurol. 2015 Feb;264:8-13). Also in the rat AAV model co- expression of C-terminal truncated alpha-synuclein enhanced full-length alpha- synuclein-induced pathology (Ulusoy et al. 2010, Eur J Neurosci. 2010 Aug ;32(3):409- 22).
• antibodies such as "GM37” and "GM285", described in the Examples
• GM37 and GM285 are capable of binding to other oligomeric forms of alpha-synuclein and altering their uptake by other CNS resident cells in a manner that reduce the spreading of disease.
• the antibodies of the invention such as GM37 and 285, were surprisingly found to be superior to prior art antibodies such as 9E4 in binding to different alpha-synuclein species in human brain, and has a surprising superior effect on clearing extracellular alpha-synuclein and normalising impaired synaptic transmission induced by the presence of abnormal alpha-synuclein in vivo. Further illustrating their therapeutic capabilities, the antibodies of the invention, such as GM37 and 285, are able to prevent the appearance of a disease related motor phenotype in a rat model for Parkinson's disease.
• antibodies GM37 and GM285 are able to inhibit seeding of aggregation and phosphorylation of endogenous alpha-synuclein induced by extracellular added recombinant pathological alpha- synuclein seeds in primary mouse neurons.
• Antibodies such as GM37 and 285 can also inhibit seeding of alpha-synuclein pathology into dopaminergic neurons in vivo using a mouse model for Parkinson's disease, further supporting the therapeutic capability of these antibodies in preventing the cell to cell propagation of pathology.
• GM37 and GM285 as new therapeutic agents capable of modifying disease through inhibition of the mechanism by which the disease pathology spreads between the neurons Parkinson's patients.
• GM37 antibody In a further aspect of the invention is provided 3 amino acid variants of the GM37 antibody. All the variants have similar functional readouts as the parent antibody, GM37, but with improved properties for manufacturability.
• the variants reduce the risk of post-translational modification occurring within the binding domain of the GM37 antibody and provide some improvement in the production of the antibody. This is advantageous because large scale clinical or commercial manufacturing of antibodies is complicated and expensive, and providing a homogenous product in pharmaceutical medicaments is crucial in particular for immunoglobulins and proteins.
• the antibodies of the invention may be used in combination with further medicaments as disclosed herein to improve the efficacy, safety and compliance of treatment
• the combinational treatments may be with symptomatic drugs such as dopamine (Pramipexole) (Loiodice et al. Psychopharmacology (Berl). 2016 Sep 10. ; Luo HT et al F. Exp Ther Med. 2016 Sep;12(3):1373-1376) or a combination with a disease modifying compound such as caspase inhibitors (e.g. prodrug VX-765;
• Pralnacasan Bassil F et al Proc Natl Acad Sci U S A. 2016 Aug 23;1 13(34) :9593-8; Wang et al Proc Natl Acad Sci U S A. 2016 Aug 23;1 13(34) :9587-92
• a Calpain inhibitor e.g. Calpeptin (ABT-957 from AbbVie) (Czapski et al FEBS Lett. 2013 Sep 17;587(18) :3135-41
• a LRRK2 inhibitor e.g. PF-06447475 (Daher et al J Biol Chem.
• a BACE1 inhibitor e.g. NB-360
• an aggregation inhibitor e.g. neuropore NPT100-18A (NPT200-1 1 ) (Wrasidio et al E. Brain. 2016 Sep 27. pii: aww238)
• EGCG Xu et al Neurochem Res. 2016
• an antibody recognising any form of ⁇ e.g. Bapineuzemab, Solanezumab, Gantenerumab, Crenezumab, Aducanumab (Guerrero-Muhoz et al Neurobiol Dis. 2014 Nov;71 :14-23; Kabiraj et al ACS Chem Neurosci. 2016 Oct 3; Hepp et al J Neuropathol Exp Neurol. 2016 Oct;75(10):936-945;Walker et al Acta Neuropathol. 2015 May;129(5):7729-48. ; an Inflammation inhibitor (e.g.
• Miglustat an inhibitor of GlcCer synthase and Eliglustat (Cerdelga) a substrate reduction therapy) (Mazzulli et al J Neurosci. 2016 Jul 20;36(29) 7693-706; Yap et al J Biol Chem. 201 1 Aug 12;286(32):28080-8; Yap et al Mol Genet Metab. 2013
• the invention relates to combinational treatments using monoclonal antibodies, and antigen-binding fragments thereof, capable of specifically binding an epitope within amino acids 1 12-1 17 in alpha-synuclein (SEQ ID NO:9 (ILEDMP)) and an additional medicament.
• the epitope bound by the antibodies or antibody-binding fragments thereof of the invention such as exemplary antibody "GM37", or "GM285", is referred to herein as "the 1 12-1 17 epitope”.
• the antibodies of the present invention specifically bind to an epitope within the 1 12-1 17 epitope and may, according to one embodiment, compete with antibody GM37 or GM285 for binding to an epitope within amino acids 112-117.
• antibodies or antigen- binding fragments thereof according to the invention may compete for binding to an epitope within amino acids 1 12-1 17 of human alpha-synuclein with a heavy chain consisting of a variable domain of SEQ ID NO:7 and a light chain consisting of a variable domain of SEQ ID NO:8.
• Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using an unlabelled binding assay such as surface plasmon resonance (SPR).
• SPR surface plasmon resonance
• a pair-wise mapping approach can be used, in which the reference antibody 'GM37' is immobilised to the surface, human alpha-synuclein antigen is bound to the immobilised antibody, and then a second antibody is tested for simultaneous binding ability to human alpha-synuclein (see 'BIAcore® Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disclosures of which are incorporated herein by reference).
• the GM285 antibody binds an epitope within residues 1 12- 1 17 of alpha-synuclein comprising residues 1 12-1 15 of alpha-synuclein (ILED; SEQ ID NO:19).
• the invention relates to combinational treatments with monoclonal antibody GM37, its variants (e.g., GM37 Variant 1 , GM37 Variant 2 and GM37 Variant 3), or GM285.
• the invention provides combinational treatments with a monoclonal antibody GM37, its variants (e.g., GM37 Variant 1 , GM37 Variant 2 and GM37 Variant 3), or GM285, and encompasses such antibodies as well as derivatives thereof that possess a sufficient number (e.g., 1 , 2, or 3) light chain CDRs and a sufficient number (e.g., 1 , 2, or 3) heavy chain CDRs to form a binding site capable of specifically binding to human synuclein.
• such antibodies will possess the three light chain CDRs and three heavy chain CDRs, as defined below.
• the numbering of amino acid residues in this region is according to IMGT®, the international
• the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
• the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
• the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
• the antibodies and antigen-binding fragments thereof of the invention may be used in combinational treatments in a method to treat synucleinopathies, such as Parkinson's disease ((PD), including idiopathic and inherited forms of Parkinson's disease), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Gauchers Disease (GD), Combined Alzheimer's and Parkinson disease (CAPD), pure autonomic failure and multiple system atrophy in combination with an additional medicament selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, aggregation inhibitors, antibodies against any form of Amyloid-beta, inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha-synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine, modulators of GCase, MAO-B inhibitors,
• synucleinopathies such as Parkinson
• the medicament may be selected from the group comprising pramipexole, VX-765 (Pralnacasan), ABT-957, PF-06447475, NB-360, Neuropore NPT100-18A, NPT200-1 1 , EGCG, Bapineuzemab, Solanezumab,
• Fig. 1 shows immunization protocols for generation of hybridomas.
• the table outlines the differences of the immunogens and mouse strains used for the
• mice Different HCo17-Balb/c and HCo12/Balb/c mice were immunized independently (description of these mice are provided below).
• the hybridoma expressing GM37 was identified from mice immunized with full length alpha- synuclein containing amino acids 1 -140 fibrils and boosted with truncated alpha- synuclein fragments 1 -60 and 1 -1 19 of full length (FL) alpha-synuclein (SEQ ID NO 10).
• the hybridoma expressing antibody GM285 came from an immunization protocol in which HCo12-Balb/c mice were immunized with full length monomeric alpha- synuclein, amino acids 1 -140 followed by a boost with full length fibrillary alpha- synuclein (Example 1 ).
• Fig. 2 shows screening of GM37 for binding to alpha synuclein, alpha-synuclein homologs and orthologs.
• Fig. 3 shows real time binding Affinity of GM37
• Goat anti-human IgG was immobilized on the CM5 chip.
• GM37 was captured on the Goat anti- human IgG immobilized chip and series of concentrations of human alpha- synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested on binding to the surface. The sensor surface was regenerated between each cycle.
• Fig. 4 shows real time binding Affinity of GM285
• Goat anti-human IgG was immobilized on the CM5 chip.
• GM285 was captured on the Goat anti-human IgG immobilized chip and series of concentrations of human alpha-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested on binding to the surface. The sensor surface was regenerated between each cycle.
• Fig. 5 shows real time binding of comparator antibody 9E4 A
• Goat anti-human IgG was immobilized on the CM5 chip.
• 9E4 was captured on the chip by its binding to Goat anti-human IgG that had been immobilized to the chip.
• a series of concentrations of human alpha-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle.
• FIG. 6 shows the amino acid sequence of alpha-synuclein.
• Major truncation sites identified by mass spectrometry in human brain tissue (Kellie JF, Higgs RE, Ryder JW, Major A, Beach TG, Adler CH, Merchant K, Knierman MD. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by mass spectrometry. Sci Rep. 2014 Jul 23;4:5797. doi: 10.1038/srep05797)
• Fig. 7 shows epitope mapping of antibody GM37 and GM285.
• ELISA data showing relative levels of binding of the antibodies to sequential peptides (20mers) derived from alpha-synuclein amino acid sequence 95 - 132 (the other nonbinding peptides are not shown).
• A) GM37 epitope requires peptide sequence ILEDMP (SEQ ID NO:9) for full binding.
• Fig. 8 shows a schematic representation of truncated forms of alpha-synuclein.
• binding epitopes of GM37/285 (ILEDMP; SEQ ID NO:9) and 9E4 (NEAYE; SEQ ID NO:36) are shown in bold on the alpha-synuclein amino acid sequence (SEQ ID NO:10). Arrows indicates the c-terminal truncations sites from Fig 6.
• alpha-synuclein A) Major truncated forms of alpha-synuclein that have been identified from human brain material. Size based on amino acid numbers is indicated on the right side. Full length alpha-synuclein is 140 amino acids. As can be deducted from the epitopes, GM37, it's variants 1 -3, and GM285 should bind full length and the 1 -1 19/122, 1 -135 fragments. Antibody 9E4 will bind only to full length and 1 -135 fragment. The specific nature of the smaller c-terminal fragments left after the truncations are not shown.
• Fig. 9 shows that antibodies GM37 and GM285 immunoprecipitate full length alpha-synuclein as well as truncated alpha-synuclein from human brain.
• Crude homogenates of human DLB brain were incubated with the test antibodies (Beads (No ab), B12-human lgG1 control antibody not binding to alpha-synuclein, GM-37, GM37 variant 2, GM-285 and murine (m)9E4) and the immunodepleted supernatant and immunoprecipitated material was separated on SDS-PAGE.
• the western blot shows the bands representing the full length and the different truncated forms of alpha- synuclein being depleted from the supernatant and being immunoprecipitated with the antibodies (IP).
• IP antibodies
• Fig. 10 shows schematics of the proteolysis of alpha-synuclein fibrils cleaved by calpain at amino acid 1 19/122.
• Alpha-synuclein fibrils (PFF) are added to the culture with (PFF+) or without (PFF) test antibody.
• PFF+ Alpha-synuclein fibrils
• PFF test antibody
• Fig. 11 A shows that GM37 inhibits the formation of the truncated band (12KD) in both the media and in cell lysates of primary mouse cortical cultures treated with PFFs. Proteins were separated by SDS-PAGE and western blotted to detect different species of alpha-synuclein. In cells treated only with PFF or the control antibody (B12) two monomeric alpha-synuclein bands are detected at 12 and 14 kDa, representing truncated and full length alpha-synuclein, respectively. In the presence of GM-37 there is only a faint band at 12Kd indicating that the majority of the cleavage is blocked. This effect is also reflected in the in the media of the cells. The relative levels of
• accumulation may also be inhibited by the presence of GM-37 as reflected in the reduction in the relative intensity of the 14Kd band. Alternatively there may be reduced amount of the 14Kd band available for uptake by the cells. (Example 5).
• Fig. 11 B shows dose dependent inhibition of proteolysis of alpha-synuclein fibrils by antibodies GM37, GM37 variant 2 and GM285.
• FL full length alpha-synulcein
• CT C-terminally truncated alpha-synuclein
• Fig. 12 shows the impact of GM37 and GM285 on seeding of alpha-synuclein aggregation and alpha-synuclein phosphorylation in mouse primary cortical neurons.
• GM37, GM37 variant 2 and GM285 inhibit appearance of phosphorylated alpha- synuclein quantitated as the number of alpha-synuclein phosphoserine 129 positive spots in cells by a Cellomics ARRAYSCANTMautomated microscope.
• GM37, GM37v2 and GM285 reduce the amount of phosphorylated alpha-synuclein spots in cells in dose dependent manner.
• Fig 13 shows basal synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic and age-matched control mice.
• Field excitatory post-synaptic potentials fEPSPs
• Basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity.
• Short-term synaptic plasticity was evaluated by induction of paired-pulse facilitation. The different intensities of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 ⁇ , and were applied successively in increasing order, with 2 to 3 repeats for each intensity.
• Basal synaptic transmission was found to be significantly impaired in F28-snca transgenic mice overexpressing wild-type alpha-synuclein compared to age-matched control mice (Example 7).
• Fig 14 shows the effect of the systemic administration of a single dose of human 9E4 (15 mg/kg, i.p.) on the impairment in basal synaptic transmission at the Schaffer collateral- CA1 synapse in the hippocampus of F28-snca transgenic mice.
• Field excitatory post-synaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity.
• Acute treatment with h9E4 induced a significant reversal of the impairment in basal synaptic
• Fig 15 shows the effect of the systemic administration of a single dose of human GM37 (15 mg/kg, i.p) or an isotype control antibody (B12) on the impairment in basal synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic mice.
• Field excitatory post-synaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity.
• Fig 16 shows the effect of the systemic administration of a single dose of human GM285 (15 mg/kg, i.p) on the impairments in basal synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic mice.
• Field excitatory post-synaptic potentials fEPSPs
• Fig 17 shows the effect of the systemic administration (15 mg/kg, i.p.) of human 9E4, GM37 or isotype control antibody (anti-HEL) on the levels of human alpha-synuclein in the interstitial fluid (isf) in the hippocampus of freely moving F28-snca transgenic mice.
• the average of the two-three basal values (4h-6h) prior to antibody treatment was taken as baseline and set to 100% for each animal. Differences were analyzed using a two-way analysis of variance (ANOVA) with repeated measures.
• the administration of GM37 induced a larger reduction in human alpha- synuclein in the hippocampus of F28 mice compared to both the comparator antibody, human 9E4, and the control isotype, anti-HEL Timepoints that show significant differences in the levels of alpha-synuclein between animals treated with GM37 or the control antibody are indicated by an asterisk. (Example 8).
• Fig 18 shows a schematic representation of the timeline for antibody treatment (down arrows), viral injections and behavioural assessment in the rat AAV human alpha-synuclein model shown in Fig. 19 (Example 9).
• Fig 19 shows that antibody GM37 can reduce Parkinsonian motor deficits after chronic treatment in the rat AAV model.
• the effect of chronic treatment with GM37 or PBS in AAV-human- alpha-synuclein rats on motor asymmetry is assessed in the cylinder test.
• Each rat was tested for the use of the forepaws by monitoring for 5 minutes.
• the percentage of use of the right forepaw (ipsilateral to the injection) and use of left (contralateral-i- right forepaws) was calculated for each animal (as shown on the y-axis) * , ** p ⁇ 0.05 and 0.01 compared to GFP-PBS rats.
• the rats treated with PBS still have a significant asymmetry in paw use, while animals treated with antibody GM37 have no longer a significant deficit. (Example 9).
• Fig 20A-20C shows that chronic treatment with antibody GM37 can reduce pathological alpha-synuclein phosphorylation induced by injection of pathological alpha-synuclein fibrillary seeds into the mouse striatum.
• Fig 20A shows a schematic indicating relative treatment times with respect to seed injection and cell counting. The antibody GM37 was administered one day before injection of recombinant alpha- synuclein fibrillary seeds into dorsal striatum of mice, and then weekly for six weeks. Dosing regimen was either 15 mg/kg iv or 30 mg/kg ip.
• Fig 20B shows the exposure level of GM37 in plasma based on site of injection and dose. Weekly samples were taken before the injection of new antibody dose.
• Fig 20C shows the exposure level of GM37 in csf based on dose and injection site at the end of the study.
• Fig 20D compares the number of cells with phosphorylated alpha-synuclein positive inclusions counted from every sixth section in substantia nigra after treatment with GM37 or PBS control.
• the mice treated with GM37 both 15 mg/kg iv and 30 mg/kg ip had a significant reduction in cells with phosphorylated alpha-synuclein inclusions compared to the PBS treated mice (Example 10).
• Fig 21 shows alignment of human a (SEQ ID NO:10), ⁇ (SEQ ID NO:37) and Y (SEQ ID NO:38) synuclein proteins. Amino acid residues different from a- synuclein are highlighted. Gaps are indicated by a dot. SwissProt numbers are in parenthesis.
• Fig 22 shows alignment of alpha-synuclein orthologs (Cynomolgus monkey, SEQ ID NO:39; Rat, SEQ ID NO:40; Mouse, SEQ ID NO:41 ). Amino acid residues different from human alpha-synuclein (SEQ ID NO:10) are highlighted. SwissProt numbers are shown in parenthesis.
• Fig 23 shows transient expression of GM37 (named GM37 wild type (wt) and 3 GM37 variants, named GM37 var 1 , 2 and 3.
• Asterisk indicates that the data are determined post protein A purification and neutralisation. ⁇ indicates that data are calculated from yield achieved post protein A and neutralization in relation to scale of expression culture (0.4L).
• Fig 24 shows a competition ELISA measuring binding of four antibodies GM37 wt, GM37 var 1 , GM37 var 2 and GM37 var 3 to human alpha-synuclein. Plates coated with alpha-synuclein are used to detect the amount of antibody remaining after preincubation in solution of each antibody (0.3 [ ⁇ g/m ⁇ ) with increasing concentration of alpha-synuclein (0-1000nM). All four antibodies show similar binding to alpha- synuclein.
• Fig 25 shows a table comparing the binding rate kinetic parameters of GM37wt and variants 1 -3 to immobilized recombinant human alpha-synuclein. The binding was measured using SPR and the rates were determined using a 1 :1 binding algorithm (BIAcore® T200).
• Fig. 26 compares the effect of alpha-synuclein antibodies on phosphorylated alpha-synuclein levels in murine primary neurons treated with pathological alpha- synuclein fibrillary seeds.
• Primary neurons were treated with seeds (1 Ong) in the presence or absence of four GM37, GM37 var 1 , GM37 var 2 and GM37 var 3 ⁇ g).
• Neurons were fixed & stained after 3 weeks and analysed by Cellomics
• Fig 27 compares temperature dependent aggregation of wt GM37, varl , var2 and var3.
• a sample of each of the antibodies was subjected to a steady increase in temperature over time and the level of aggregation was simultaneously measured by multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies).
• the temperature for onset of aggregation is similar for GM37 and GM37-variants, however the lowest level of aggregation observed for GM37-Var2.
• alpha-synuclein is synonymous with “the alpha- synuclein protein” and refers to any of the alpha-synuclein protein isoforms (identified in, for example, UniProt as P37840, 1 -3).
• the amino acid numbering of alpha-synuclein is given with respect to SEQ ID NO:10 as shown below, with methionine (M) being amino acid residuel :
• the present invention relates to antibodies and to fragments of antibodies that are capable of specifically binding to alpha-synuclein, and in particular to human alpha- synuclein.
• the antibodies and fragments thereof exhibit the ability to specifically bind to an epitope within 1 12-1 17 of human alpha-synuclein.
• antibody in the context of the present invention refers to an immunoglobulin molecule or according to some embodiments of the invention, a fragment of an immunoglobulin molecule which has the ability to specifically bind to an epitope of a molecule ("antigen").
• Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, usually comprised of three domains (CH1 , CH2 and CH3).
• Heavy chains can be of any isotype, including IgG (lgG1 , lgG2, lgG3 and lgG4 subtypes), IgA (lgA1 and lgA2 subtypes), IgM and IgE.
• Each light chain is comprised of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL).
• Light chains include kappa chains and lambda chains.
• the heavy and light chain variable domain is typically responsible for antigen recognition, while the heavy and light chain constant domain may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system.
• VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions,” that are interspersed with regions of more conserved sequence, termed “framework regions” (FR).
• FR frame regions
• Each VH and VL is composed of three CDR Domains and four FR Domains arranged from amino-terminus to carboxy-terminus in the following order: FR1 -CDR1 -FR2-CDR2- FR3-CDR3-FR4.
• the variable domains of the heavy and light chains contain a binding domain that interacts with an antigen.
• antibodies and their antigen-binding fragments that have been "isolated” so as to exist in a physical milieu distinct from that in which it may occur in nature or that have been modified so as to differ from a naturally occurring antibody in amino acid sequence.
• epitope means an antigenic determinant capable of specific binding to an antibody.
• Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and linear epitopes are distinguished in that the binding to the former, but not the latter, is always lost in the presence of denaturing solvents.
• the epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide).
• 1 12-1 17 epitope refers to a region of human alpha-synuclein that contains at least 4 of the 6 amino acid residues of 1 12-1 17 human alpha-synuclein, which epitope does not include any residue from 1 -1 1 1 (including any residue from 106-1 1 1 ) of human alpha-synuclein, nor any residue from 1 18-140 (including residue 1 18-120) of human alpha-synuclein.
• an antibody is said to be capable of specifically binding to an epitope within the "1 12-1 17 epitope” if it is capable of specifically binding to human alpha-synuclein by binding to at least 4 of the 6 amino acid residues of the 1 12-1 17 epitope.
• the term "antigen-binding fragment of an antibody” means a fragment, portion, region or domain of an antibody (regardless of how it is produced (e.g., via cleavage, recombinantly, synthetically, etc.)) that is capable of specifically binding to an epitope, and thus the term “antigen-binding” is intended to mean the same as “epitope-binding” so that, for example, an "antigen-binding fragment of an antibody” is intended to be the same as an “epitope-binding fragment of an antibody”.
• An antigen-binding fragment may contain 1 , 2, 3, 4, 5 or all 6 of the CDR Domains of such antibody and, although capable of specifically binding to such epitope, may exhibit a specificity, affinity or selectivity toward such epitope that differs from that of such antibody. Preferably, however, an antigen-binding fragment will contain all 6 of the CDR Domains of such antibody.
• An antigen-binding fragment of an antibody may be part of, or comprise, a single polypeptide chain (e.g., an scFv), or may be part of, or comprise, two or more polypeptide chains, each having an amino-terminus and a carboxyl terminus (e.g., a diabody, a Fab fragment, a Fab 2 fragment, etc.).
• Fragments of antibodies that exhibit antigen-binding ability can be obtained, for example, by protease cleavage of intact antibodies. More preferably, although the two domains of the Fv fragment, VL and VH, are naturally encoded by separate genes, or polynucleotides that encode such gene sequences (e.g., their encoding cDNA) can be joined, using recombinant methods, by a flexible linker that enables them to be made as a single protein chain in which the VL and VH regions associate to form monovalent antigen-binding molecules (known as single-chain Fv (scFv) ; see e.g., Bird et al. , (1988) Science 242:423-426; and Huston et al.
• scFv single-chain Fv
• a bispecific antibody, diabody, or similar molecule in which two such polypeptide chains associate together to form a bivalent antigen- binding molecule
• antigen-binding fragments encompassed within the present invention include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in
• WO2007059782 F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge domain; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of a VL and VH domains, (v) a dAb fragment (Ward et al., Nature 341 , 544- 546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol.
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)).
• scFv single chain antibodies or single chain Fv
• antibody also includes antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.
• An antibody as generated can possess any isotype.
• isotype refers to the immunoglobulin class (for instance lgG1 , lgG2, lgG3 or lgG4) that is encoded by heavy chain constant domain genes.
• bispecific antibody refers to an antibody containing two independent antigen-binding fragments that each target independent targets. These targets can be epitopes present on different proteins or different epitopes present on the same target.
• Bispecific antibody molecules can be made using compensatory amino acid changes in the constant domains of the HCs of the parent monospecific bivalent antibody molecules. The resulting heterodimeric antibody contains one Fabs contributed from two different parent monospecific antibodies. Amino acid changes in the Fc domain leads to increased stability of the heterodimeric antibody with bispecificity that is stable over time.
• Bispecific antibodies can also include molecules that are generated using ScFv fusions.
• Bispecific molecules Two monospecific scfv are then independently joined to Fc domains able to form stable heterodimers to generate a single bispecific molecule (Mabry et al., PEDS 23:3 1 15-127 (2010).
• Bispecific molecules have dual binding capabilities. For example, targeting both a therapeutic target and a transcytosing surface receptor for the purpose of delivering a therapeutic antibody across the blood brain barrier to treat a CNS disease.
• GM37, GM-37, GM37 wild type (wt), mab37 and 6004-37 are used interchangeably herein and all refer to the same antibody.
• antibody GM37 is intended to include an antibody or antigen-binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1 -3 SEQ ID Nos:1-3 and the Light Chain CDR1 -3 as given in SEQ ID Nos:4-6.
• the antibody GM37 or antigen-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:7 and/or the light chain variable domain of SEQ ID NO:8.
• the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:7 and a constant domain of SEQ ID NO:18 together with a light chain consisting of a variable domain of SEQ ID NO:8 and a kappa constant domain of SEQ ID NO:17.
• Deamination of proteins, and in these instance antibodies can occur spontaneously during manufacturing and storage, but also in vivo, and makes the quality of the final pharmaceutical medicament difficult to control.
• the deamination may also in some instances affect the activity of the molecule. Deamination occurs at asparagine residues, but the location of the relevant asparagine may be difficult to predict with certainty, but may be influenced in some instances by an asparagine- glycine motif.
• Several possible deamination motifs are found on the GM37 antibody, however, one likely site of deamination was found to be at residue 54 of the heavy chain.
• the subsequent substitution of asparagine by another amino acid is not straight forward, but 3 variants of GM37 (GM37 variant (var) 1 , 2 and 3) were found to retain the activity of the original GM37 (GM37 wild type (wt)).
• GM37 variants refers to the deaminated variants 1 ,2 or 3, wherein variant 1 has a N54S substitution, variant 2 has a N54Q substitution and variant 3 has a N54H compared to the GM37 antibody described herein above.
• the antibody GM37 variant (var) 1 , 2 and 3 are thus intended to include an antibody or antigen-binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1 and 3 SEQ ID Nos:1 and 3 from GM 37 and the Light Chain CDR1 -3 from GM37 as given in SEQ ID Nos:4-6, but differing in their heavy chain CDR2 so that variant 1 has CDR 2 of SEQ ID NO:33, variant 2 has CDR 2 of SEQ ID NO:34 and variant 3 has CDR 2 of SEQ ID NO:35.
• the antibody GM37 variants or their antigen-binding fragments may comprise or consist of the heavy chain variable domain of SEQ ID NO:30, 31 and 32 for variant 1 , 2 and 3, respectively, and the light chain variable domain of SEQ ID NO:8.
• the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:30, 31 or 32 and a constant domain of SEQ ID NO:18 together with a light chain consisting of a variable domain of SEQ ID NO:8 and a kappa constant domain of SEQ ID NO:17.
• the terms GM285, GM-285, mab285 and 6004-285 are used interchangeably herein and all refer to the same antibody.
• antibody GM285 is intended to include an antibody or antigen- binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1 -3 SEQ ID NOs:20-22 and the Light Chain CDR1 -3 as given in SEQ ID NOs:23- 25.
• the antibody GM37 or antigen-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:26 and/or the light chain variable domain of SEQ ID NO:27.
• the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:26 and a constant domain of SEQ ID NO:28 together with a light chain consisting of a variable domain of SEQ ID NO:27 and a kappa constant domain of SEQ ID NO:29.
• the GM285 antibody specifically binds an epitope within the sequence 1 12- 1 15 (ILED; SEQ ID NO:19) of human alpha-synuclein (SEQ ID NO:10).
• IMGT® the international ImMunoGeneTics information system® or, Kabat, E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S. & Foeller, C.
• an "anti-alpha-synuclein antibody” or “alpha-synuclein antibody” is an antibody or an antigen-binding fragment thereof which binds specifically to alpha-synuclein or an alpha-synuclein fragment as defined herein above, in particular the sequence of alpha- synuclein corresponding to SEQ ID NOs 9 and/or 19.
• the term "human antibody” (which may be abbreviated to "humAb” or “HuMab”), as used herein, is intended to include antibodies having variable and constant domains derived from human germline immunoglobulin sequences.
• the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
• the term "combined” as used herein in the context of the method/use of the invention comprises the administration of therapeutically effective amounts of 1 ) an alpha- synuclein antibody according to the invention and 2) a compound useful for the treatment of of Parkinson's disease (including idiopathic and inherited forms of Parkinson's disease), Gauchers Disease (GD), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson's disease, pure autonomic failure and multiple system atrophy.
• Parkinson's disease including idiopathic and inherited forms of Parkinson's disease
• GD Gauchers Disease
• DLBD Diffuse Lewy Body Disease
• LBV Lewy body variant of Alzheimer's
• the administration of the 2 medicaments may occur simultaneously or separately in therapeucally effective amounts to achive the desired treatment effect of the combined treatment.
• additional medicaments may be selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, aggregation inhibitors, antibodies recognising any form of Amyloid-beta, inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha-synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine, modulators of GCase, MAO-B inhibitors, Levodopa/carbidopa, Dopamine agonists, COMT inhibitors and A2A antagonists to said subject in an effective amount.
• the additional medicaments are is selected from the group comprising pramipexole, VX-765 (Pralnacasan), ABT-957, PF- 06447475, NB-360, Neuropore NPT100-18A, NPT200-1 1 , EGCG, Bapineuzemab, Solanezumab, Gantenerumab, Crenezumab, Aducanumab, C16H15N04, BMS-986016, GSK2831781 , IMP321 , ELN484228, NCGC607, Miglustat, Eliglustat (Cerdelga), Azilect, Eldepryl, Zelapar, Pramipexole, Ropinerole, Bromocriptine, Comtess, Tasmar and Istradefyline.
• the compounds used in the combinational treatment may be administered simultaneously or with a time gap between the administrations of the two compounds.
• the two compounds may be administered on the same day or on different days or time intervals.
• the terms "monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
• a conventional monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
• a monoclonal antibody can be composed of more than one Fab domain thereby increasing the specificity to more than one target.
• the terms “monoclonal antibody” or “monoclonal antibody composition” are not intended to be limited by any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).
• the term “humanized” refer to a molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an
• the antigen- binding site may comprise either complete non-human antibody variable domains fused to human constant domains, or only the complementarity determining regions (CDRs) of such variable domains grafted to appropriate human framework regions of human variable domains.
• the framework residues of such humanized molecules may be wild type (e.g., fully human) or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence has served as the basis for humanization. Humanization lessens or eliminates the likelihood that a constant domain of the molecule will act as an immunogen in human individuals, but the possibility of an immune response to the foreign variable domain remains
• variable domains of both heavy and light chains contain three complementarity- determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
• CDRs complementarity- determining regions
• FRs framework regions
• humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
• humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.
• an antibody or an antigen-binding fragment thereof is said to "specifically" bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity or avidity with that epitope relative to alternative epitopes.
• the antibody, or antigen-binding fragment thereof, of the invention binds at least 10-fold more strongly to its target (human alpha synuclein) than to another molecule;
• the antibody, or antigen-binding fragment thereof binds under physiological conditions, for example, in vivo.
• an antibody that is capable of "specifically binding" to an epitope within residues 1 12-1 17 (ILEDMP (SEQ ID NO:9)) of human alpha-synuclein encompasses an antibody or antigen-binding fragments thereof, that is capable of binding to an epitope within residues 1 12-1 17 of human alpha-synuclein with such specificity and/or under such conditions.
• binding in the context of the binding of an antibody to a predetermined antigen typically refers to binding with an affinity corresponding to a KD of about 10 7 M or less, such as about 10 -8 M or less, such as about 10 -9 M or less when determined by for instance surface plasmon resonance (SPR) technology in either a BIAcore® 3000 or T200instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1 ,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
• a non-specific antigen e.g., BSA, casein
• the amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.
• kd (sec -1 or 1/s), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
• ka (M-1 x sec-1 or 1/Msec), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
• KD (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the kd by the ka.
• KA (M-1 or 1/M), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.
• the invention relates to an antibody or antigen-binding fragments thereof, which exhibits one or more of the following properties:
• KD binding affinity for alpha-synuclein of between 0.5-10 nM, such as 1 -5 nM or 1 -2 nM;
• binding affinity (KD) for alpha-synuclein may be determined using methods well known in the art, e.g. as described in Example 2.
• the term "capability of inhibiting protease truncation of alpha-synuclein fibrils" includes the capability of inhibiting calpain-1 induced formation of fragment 1 -1 19-122 of human alpha synuclein in primary cortical neurons (see Example 5).
• the term "capability of reversing impairment in basal synaptic transmission in F28-snca transgenic mice” includes the capability of reverse the impairment of synaptic transmission and plasticity in the CA1 area of the hippocampus in F28-snca transgenic mice, for example as indicated by evoked fEPSP slope as measured
• the term "capability of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis” includes the capability of reducing levels of human alpha synuclein in the hippocampus awake, freely-moving F28-snca transgenic mice, as measured using in vivo microdialysis (see Example 7).
• the term "capability, when administered chronically, to restore motor function in a rat model of Parkinson's disease” include the capability to reduce or eliminate motor asymmetry in a rat recombinant adeno-associated viral vector (rAAV) model of Parkinson's Disease (see Example 8).
• CDR residues not contacting the relevant epitope and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (see, Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL I NTEREST, National Institutes of Health Publication No. 91 -3242; Chothia, C. et al. (1987)
• substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
• a polynucleotide encoding the CDR is mutagenized (for example via random mutagenesis or by a site-directed method (e.g., polymerase chain-mediated amplification with primers that encode the mutated locus)) to produce a CDR having a substituted amino acid residue.
• a site-directed method e.g., polymerase chain-mediated amplification with primers that encode the mutated locus
• the BLOSUM62.iij substitution score for that substitution can be identified.
• the BLOSUM system provides a matrix of amino acid substitutions created by analyzing a database of sequences for trusted alignments (Eddy, S.R.
• BLOSUM62.iij the BLOSUM62 database
• Table 1 presents the BLOSUM62.iij substitution scores (the higher the score the more conservative the substitution and thus the more likely the substitution will not affect function). If an antigen-binding fragment comprising the resultant CDR fails to bind to alpha-synuclein, for example, then the BLOSUM62.iij substitution score is deemed to be insufficiently conservative, and a new candidate substitution is selected and produced having a higher substitution score.
• the BLOSUM62.iij substitution score will be 0, and more conservative changes (such as to aspartate, asparagine, glutamine, or lysine) are preferred.
• the invention thus contemplates the use of random mutagenesis to identify improved CDRs.
• conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
• More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
• Phage display technology can alternatively be used to increase (or decrease) CDR affinity.
• This technology referred to as affinity maturation, employs mutagenesis or "CDR walking" and re-selection uses the target antigen or an antigenic antigen- binding fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations.
• Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue.
• Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased affinity to the antigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci. (U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking which randomizes the Light Chain may be used possible (see, Schier et al., 1996, J. Mol. Bio. 263:551 ).
• the sequence of CDR variants of encompassed antibodies or their antigen-binding fragments may differ from the sequence of the CDR of the parent antibody, GM37, GM37 var 1 -3, or 285, through substitutions; for instance substituted 4 amino acid residue, 3 amino acid residue, 2 amino acid residue or 1 of the amino acid residues.
• substitutions for instance substituted 4 amino acid residue, 3 amino acid residue, 2 amino acid residue or 1 of the amino acid residues.
• the amino acids in the CDR regions may be substituted with conservative substitutions, as defined in the 3 tables above.
• treatment means ameliorating, slowing, attenuating or reversing the progress or severity of a disease or disorder, or ameliorating, slowing, attenuating or reversing one or more symptoms or side effects of such disease or disorder.
• treatment or “treating” further means an approach for obtaining beneficial or desired clinical results, where "beneficial or desired clinical results” include, without limitation, alleviation of a symptom, diminishment of the extent of a disorder or disease, stabilized (i.e., not worsening) disease or disorder state, delay or slowing of the progression a disease or disorder state, amelioration or palliation of a disease or disorder state, and remission of a disease or disorder, whether partial or total, detectable or undetectable.
• an "effective amount,” when applied to an antibody or antigen-binding fragments thereof, of the invention, refers to an amount sufficient, at dosages and for periods of time necessary, to achieve an intended biological effect or a desired therapeutic result including, without limitation, clinical results.
• therapeutically effective amount when applied to an antibody or antigen-binding fragments thereof, of the invention is intended to denote an amount of the antibody, or antigen-binding fragment thereof, that is sufficient to ameliorate, palliate, stabilize, reverse, slow, attenuate or delay the progression of a disorder or disease state, or of a symptom of the disorder or disease.
• the method of the present invention provides for administration of the antibody, or antigen-binding fragment thereof, in combinations with other compounds.
• the "effective amount” is the amount of the combination sufficient to cause the intended biological effect.
• a therapeutically effective amount of an anti-alpha-synuclein antibody or antigen-binding fragment thereof of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti- alpha-synuclein antibody to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
• the present invention particularly relates to a monoclonal antibody capable of specifically binding to an epitope within amino acids 1 12-1 17 of human alpha-synuclein (SEQ ID NO:9 (ILEDMP)).
• the antibody is capable of competing with the antibody GM37 for binding to an epitope within the 1 12-1 17 amino acids of alpha-synuclein.
• the antibodies of the present invention exemplified by GM37 its variants GM37 var 1 -3 and GM285, and their alpha-synuclein binding fragments are capable of binding the toxic alpha-synuclein fragment consisting of residues 1 -1 19/122 of alpha-synuclein and neutralizing its toxicity (for example, by extracellular binding to the alpha-synuclein fragment and thereby preventing it from being taken up by cells.
• the antibodies of the present invention which are capable of binding to an epitope within amino acids 1 12-1 17 of alpha-synuclein are superior to prior art antibodies such as antibody 9E4 in binding to toxic alpha-synuclein species in human brain, and have superior effects on clearing extracellular alpha-synuclein and normalising an impaired synaptic transmission induced by alpha-synuclein in vivo.
• the antibodies of the invention are also able to ameliorate the appearance of a relevant motor phenotype in a rat model for Parkinson's disease.
• the antibodies of the present invention are preferably human or humanized antibodies.
• the present invention also provides a method of reducing alpha- synuclein aggregate formation in a patient, comprising administering to the patient in need of such treatment, a therapeutically effective amount of an antibody of the invention.
• the antibodies may be administered together, either simultationsly or separately, with another medicament selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, aggregation inhibitors, antibodies recognising any form of Amyloid-beta, inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha-synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine, modulators of GCase, MAO-B inhibitors, Levodopa/carbidopa, Dopamine agonists, COMT inhibitors and A 2 A antagonists.
• These medicaments may specifically be selected from the group comprising pramipexole, VX-765 (Pralnacasan), ABT-957, PF-06447475, NB-360, Neuropore NPT100-18A, NPT200-1 1 , EGCG, Bapineuzemab, Solanezumab,
• GSK2831781 IMP321 , ELN484228, NCGC607, Miglustat, Eliglustat (Cerdelga), Azilect, Eldepryl, Zelapar, Pramipexole, Ropinerole, Bromocriptine, Comtess, Tasmar and Istradefyline.
• the antibodies may be in a composition together with a pharmaceutically acceptable carrier, diluent and/or stabilizer.
• the antibodies of the invention may be used in therapy.
• the antibodies of the invention may be used in treating synucleinopathies such as Parkinson's disease (including idiopathic inherited forms of Parkinson's disease), Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined
• synucleinopathies such as Parkinson's disease (including idiopathic inherited forms of Parkinson's disease), Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined
• Alzheimer's and Parkinson disease pure autonomic failure and multiple system atrophy.
• the treatment envisioned by the present invention may be chronic and the patient may be treated at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.
• the antibodies of antigen-binding fragments thereof of the present invention may be produced in different cell lines, such as a human cell line, a mammal non-human cell line, and insect cell line, for example a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18:1 -13., the contents which is included herein by reference).
• insect cell line for example a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18
• the antibodies of the present invention may for example be monoclonal antibodies produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991 ) and Marks et al., J. Mol. Biol. 222, 581 -597 (1991 ). Monoclonal antibodies may be obtained from any suitable source.
• monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B lymphocyte cells obtained from mice immunized with an antigen of interest, for instance, in the form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest.
• Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, rabbits, dogs, sheep, goats, primates, etc.
• the antibody of the invention is a human antibody.
• Human monoclonal antibodies directed against alpha-synuclein may be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
• transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively.
• the HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged human heavy variable and constant ( ⁇ and Y) and light variable and constant ( ⁇ ) chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or IgK and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG, ⁇ monoclonal antibodies (Lonberg, N. et al.
• HuMAb mice were prepared in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992) , Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L.
• mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 81 1 -820 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)).
• kappa endogenous light chain
• CMD disruption in their endogenous heavy chain genes as described in Example 1 of WO 01/14424
• KCo5 human kappa light chain transgene as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).
• the HCo7 mice have a HCo7 human heavy chain transgene (as described in US 5,770,429)
• the HCo12 mice have a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424)
• the HCo17 mice have a HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187)
• the HCo20 mice have a HCo20 human heavy chain transgene.
• the resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
• the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 81 1 -820
• mice (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187.
• This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).
• This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478.
• HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can be generated by crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) as described in WO 09/097006.
• the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 81 1 -820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187.
• This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).
• This mouse strain also carries a human heavy chain trans-chromosome composed of chromosome 14 antigen-binding fragment hCF (SC20) as described in WO 02/43478.
• Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.
• Human monoclonal or polyclonal antibodies of the present invention, or antibodies of the present invention originating from other species may also be generated transgenically through the generation of another non-human mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom.
• antibodies may be produced in, and recovered from, the milk of goats, cows, or other mammals. See for instance US 5,827,690, US 5,756,687, US 5,750,172 and US 5,741 ,957.
• the antibody of the invention may be of any isotype.
• the choice of isotype typically will be guided by the desired effector functions, such as ADCC induction.
• Exemplary isotypes are lgG1 , lgG2, lgG3, and lgG4. Either of the human light chain constant domains, kappa or lambda, may be used.
• the class of an anti-alpha-synuclein antibody of the present invention may be switched by known methods. For example, an antibody of the present invention that was originally IgM may be class switched to an IgG antibody of the present invention. Further, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGI to lgG2.
• an antibody of the present invention is an lgG1 antibody, for instance an lgG1 , ⁇ .
• An antibody is said to be of a particular isotype if its amino acid sequence is most homologous to that isotype, relative to other isotypes.
• the antibody of the invention is a full-length antibody, preferably an IgG antibody, in particular an lgG1 , ⁇ antibody.
• the antibody of the invention is an antibody fragment or a single-chain antibody.
• Antibodies and antigen-binding fragments thereof may e.g. be obtained by antigen-binding fragmentation using conventional techniques, and antigen-binding fragments screened for utility in the same manner as described herein for whole antibodies.
• F(ab')2 antigen-binding fragments may be generated by treating antibody with pepsin.
• the resulting F(ab')2 antigen-binding fragment may be treated to reduce disulfide bridges to produce Fab' antigen-binding fragments.
• Fab antigen-binding fragments may be obtained by treating an IgG antibody with papain; Fab' antigen-binding fragments may be obtained with pepsin digestion of IgG antibody.
• An F(ab') antigen-binding fragment may also be produced by binding Fab'-described below via a thioether bond or a disulfide bond.
• a Fab' antigen-binding fragment is an antibody antigen-binding fragment obtained by cutting a disulfide bond of the hinge domain of the F(ab')2.
• a Fab'- antigen-binding fragment may be obtained by treating an F(ab')2 antigen-binding fragment with a reducing agent, such as dithiothreitol.
• Antibody antigen-binding fragment may also be generated by expression of nucleic acids encoding such antigen-binding fragments in recombinant cells (see for instance Evans et al., J. Immunol. Meth.
• a chimeric gene encoding a portion of an F(ab')2 antigen-binding fragment could include DNA sequences encoding the CH1 region and hinge domain of the H chain, followed by a translational stop codon to yield such a truncated antibody antigen-binding fragment molecule.
• the anti-alpha-synuclein antibody is a monovalent antibody, preferably a monovalent antibody as described in WO2007059782 (which is incorporated herein by reference in its entirety) having a deletion of the hinge domain.
• the antibody is a monovalent antibody
• said anti-alpha-synuclein antibody is constructed by a method comprising: i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific anti-alpha-synuclein antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an lgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG
• the anti- alpha-synuclein antibody is a monovalent antibody, which comprises:
• a CH domain of an immunoglobulin or a domain thereof comprising the CH2 and CH3 domains, wherein the CH domain or domain thereof has been modified such that the domain corresponding to the hinge domain and, if the immunoglobulin is not an lgG4 subtype, other domains of the CH domain, such as the CH3 domain, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH domain or other covalent or stable non-covalent inter- heavy chain bonds with an identical CH domain in the presence of polyclonal human IgG.
• the heavy chain of the monovalent anti-alpha- synuclein antibody has been modified such that the entire hinge domain has been deleted.
• the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.
• the invention also includes "Bispecific Antibodies," wherein an anti- Alpha-synuclein binding region (e.g., a Alpha-synuclein-binding region of an anti-alpha- synuclein monoclonal antibody) is part of a bivalent or polyvalent bispecific scaffold that targets more than one epitope, (for example a second epitope could comprise an epitope of an active transport receptor, such that the Bispecific Antibody would exhibit improved transcytosis across a biological barrier, such as the Blood Brain Barrier).
• the monovalent Fab of an anti-synuclein antibody may be joined to an additional Fab or scfv that targets a different protein to generate a bispecific antibody.
• a bispecific antibody can have a dual function, for example a therapeutic function imparted by an anti-synuclein binding domain and a transport function that can bind to a receptor molecule to enhance transfer cross a biological barrier, such as the blood brain barrier.
• Anti-alpha-synuclein antibodies, and antigen-binding fragments thereof, of the invention also include single chain antibodies.
• Single chain antibodies are peptides in which the heavy and light chain Fv regions are connected.
• the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-alpha-synuclein antibody of the present invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain.
• a flexible peptide linker typically of about 10, 12, 15 or more amino acid residues
• the single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.
• the anti-alpha-synuclein antibodies and antigen-binding fragments thereof described herein may be modified by inclusion of any suitable number of modified amino acids and/or associations with such conjugated substituents.
• Suitability in this context is generally determined by the ability to at least substantially retain the alpha-synuclein selectivity and/or the anti-alpha-synuclein specificity associated with the non-derivatized parent anti-alpha-synuclein antibody.
• the inclusion of one or more modified amino acids may be advantageous in, for example, increasing polypeptide serum half-life, reducing polypeptide antigenicity, or increasing polypeptide storage stability.
• Amino acid(s) are modified, for example, co-translationally or post- translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means.
• Non- limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e. g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
• the modified amino acid may, for instance, be selected from a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent.
• Anti-alpha-synuclein antibodies may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546.
• Additional illustrative polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1 ,000 and about 40,000, such as between about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol).
• PEG polyethylene glycol
• the antibodies of antigen-binding fragments thereof of the present invention may be produced in different cell lines, such as a human cell line, a mammal non-human cell line, and insect cell line, for example a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18:1 -13., the contents which is included herein by reference).
• insect cell line for example a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18
• the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody or an antigen-binding domain thereof of the invention as defined herein or a bispecific molecule of the invention as defined herein.
• host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells.
• the present invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an anti-alpha-synuclein antibody of the present invention or an antigen-binding fragment thereof.
• the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-alpha-synuclein antibody of the invention.
• a non-integrated nucleic acid such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-alpha-synuclein antibody of the invention.
• the invention relates to a preparation that, as such term is used herein, comprises an anti-alpha-synuclein antibody as defined herein, and that is substantially free of naturally-arising antibodies that are either not capable of binding to alpha-synuclein or that do not materially alter the anti-alpha-synuclein functionality of the preparation.
• a preparation does not encompass naturally-arising serum, or a purified derivative of such serum, that comprises a mixture of an anti-alpha-synuclein antibody and another antibody that does not alter the functionality of the anti-alpha-synuclein antibody of the preparation; wherein such functionality is selected from the group consisting of:
• a capability to prevent seeding of alpha-synuclein such as accumulation of insoluble phosphorylated alpha-synuclein in vitro and/or in a mouse model of Parkinson's disease.
• the invention particularly relates to preparations of such an anti-alpha- synuclein antibody having a structural change in its amino acid sequence (in any of its CDRs, variable domains, framework residues and/or constant domains) relative to the structure of a naturally-occurring anti-alpha-synuclein antibody, wherein said structural change causes the anti-alpha-synuclein monoclonal antibody to exhibit a markedly altered functionality (i.e., more than a 20% difference, more than a 40% difference, more than a 60% difference, more than an 80% difference, more than a 100% difference, more than a 150% difference, more than a 2-fold difference, more than a 4- fold difference, more than a 5-fold difference, or more than a 10-fold difference in functionality) relative to the functionality exhibited by said naturally-occurring anti- alpha-synuclein antibody; wherein such functionality is:
• substantially free of naturally-arising antibodies refers to the complete absence of such naturally-arising antibodies in such preparations, or of the inclusion of a concentration of such naturally-arising antibodies in such preparations that does not materially affect the alpha-synuclein-binding properties of the
• preparations refers to antibodies (including naturally-arising autoantibodies) elicited within living humans or other animals, as a natural consequence to the functioning of their immune systems.
• the preparations of the present invention do not exclude, and indeed explicitly encompass, such preparations that contain an anti-alpha-synuclein antibody and a deliberately added additional antibody capable of binding to an epitope that is not possessed by alpha-synuclein.
• Such preparations particularly include embodiments thereof wherein the preparation exhibits enhanced efficacy in treating synucleinopathies such as Parkinson's disease (including idiopathic and inherited form of Parkinson's disease), Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomic failure and multiple system atrophy.
• synucleinopathies such as Parkinson's disease (including idiopathic and inherited form of Parkinson's disease), Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomic failure and multiple system atrophy.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising:
• compositions may be formulated with
• compositions should be suitable for the chosen compound of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on epitope binding.
• a pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., a non-ionic detergent, such as Tween- 20 or Tween- 80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
• the diluent is selected to not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
• compositions or formulation may also include other carriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers and the like.
• the compositions may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides like chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).
• the actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• the pharmaceutical composition may be administered by any suitable route and mode, including: parenteral, topical, oral or intranasal means for prophylactic and/or therapeutic treatment.
• a pharmaceutical composition of the present invention is administered parenterally.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion. Additional suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art. In one embodiment that pharmaceutical composition is administered by intra
• Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are
• aqueous and non-aqueous carriers examples include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers.
• Other carriers are well known in the pharmaceutical arts.
• Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for
• compositions of the present invention are known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. [000145] Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
• oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butyl
• compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
• isotonicity agents such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
• compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition.
• adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition.
• the compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
• the compounds of the present invention may be formulated to ensure proper distribution in vivo.
• Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
• compositions for injection must typically be sterile and stable under the conditions of manufacture and storage.
• the composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration.
• the carrier may be an aqueous or non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
• the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays antibody absorption, for example, monostearate salts and gelatin.
• Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above.
• sterile powders for the preparation of sterile injectable solutions examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Dosage regimens in the above methods of treatment and uses described herein are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
• compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
• the effective dosages and the dosage regimens for the anti alpha- synuclein antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. On any given day that a dosage is given, the dosage may range from about 0.0001 to about 100 mg/kg, and more usually from about 0.01 to about 5 mg/kg, of the host body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1 -10 mg/kg body weight.
• Exemplary dosages thus include: from about 0.1 to about 10 mg/kg/body weight, from about 0.1 to about 5 mg/kg/body weight, from about 0.1 to about 2 mg/kg/body weight, from about 0.1 to about 1 mg/kg/body weight, for instance about 0.15 mg/kg/body weight, about 0.2 mg/kg/body weight, about 0.5 mg/kg/body weight, about 1 mg/kg/body weight, about 1 .5 mg/kg/body weight, about 2 mg/kg/body weight, about 5 mg/kg/body weight, or about 10 mg/kg/body weight.
• a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
• the physician could start doses of the anti-alpha-synuclein antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
• a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
• Such an effective dose will generally depend upon the factors described above.
• Administration may e.g. be intravenous, intramuscular,
• composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition as described above.
• the invention relates to an antibody or antigen- binding fragments thereof, of the invention, for use in medicine.
• the invention relates to an antibody or antigen- binding fragments thereof, of the invention, for use in treating a synucleinopathy in combination with another medicament selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE inhibitors, aggregation inhibitors, ⁇ antibodies, inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha-synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine and modulators of GCase, MAO-B inhibitors,
• the medicament may be selected from the group comprising pramipexole, VX-765 (Pralnacasan), ABT-957, PF-06447475, NB-360, Neuropore NPT100-18A, NPT200-1 1 , EGCG, Bapineuzemab, Solanezumab,
• the monoclonal antibody, or antigen-binding fragment thereof, together with an additional medicament refered to herein above is for use in treating Parkinson's disease, idiopathic Parkinson's disease, familiar forms of Parkinson's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson's disease, pure autonomic failure or multiple system atrophy.
• Parkinson's disease idiopathic Parkinson's disease
• DLBD Diffuse Lewy Body Disease
• LBV Lewy body variant of Alzheimer's disease
• Combined Alzheimer's and Parkinson's disease pure autonomic failure or multiple system atrophy.
• the invention relates to the use of the antibody, or antigen-binding fragment thereof, of the invention together with an additional medicament mentioned herein above, in the manufacture of a medicament for treating a synucleinopathy.
• the invention relates to the use of an antibody of the invention for the preparation of a medicament for use in treatment of Parkinson's disease (including idiopathic and inherited forms of Parkinson's disease), Gauchers Disease (GD), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson's disease, pure autonomic failure and multiple system atrophy wherein the patient is treated with an additional medicament selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE inhibitors, aggregation inhibitors, antibodies against ⁇ , inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha- synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine and modulators of GCase, MAO-B inhibitors, Levodopa/carbidopa, Dopamine agonists, COMT inhibitors and A
• pramipexole VX-765 (Pralnacasan), ABT-957, PF-06447475, NB-360, Neuropore NPT100-18A, NPT200-1 1 , EGCG, Bapineuzemab, Solanezumab, Gantenerumab, Crenezumab, Aducanumab, C16H15N04, BMS-986016, GSK2831781 , IMP321 , ELN484228, NCGC607, Miglustat, Eliglustat (Cerdelga), Azilect, Eldepryl, Zelapar, Pramipexole, Ropinerole, Bromocriptine, Comtess, Tasmar and Istradefyline.
• the invention relates to a treating Parkinson's disease or other synucleinopathies, comprising administering an effective dosage of an antibody of the invention, or an antigen-binding fragment thereof together with an effective therapeutic amount of another medicament mentioned herein above.
• the treatment is chronic, and is preferably for at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.
• kits comprising an antibody according to the invention and a medicaments selected from the group comprising dopamine, caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE inhibitors, aggregation inhibitors, antibodies against ⁇ , inflammation inhibitors, LAG-3 antibodis, Isradipine, molecules reversing alpha-synuclein induced reduction in phagocytic activity of microglia, nicotine, caffeine and modulators of GCase, MAO-B inhibitors,
• kits may in antother embodment contain an antibody according to the inventionea second medicament selected from the group comprising pramipexole, VX-765
• the kit may for the use in treating Parkinson's disease), Gauchers
• GD Diffuse Lewy Body Disease
• LBD Lewy body variant of Alzheimer's disease
• DLBD Diffuse Lewy Body Disease
• LBV Lewy body variant of Alzheimer's disease
• Example 1 Antibody Screening 1. Immunogen and ligand production
• Fig 1 The mice were immunized with three immunogens: full length recombinant human alpha-synuclein fibrils; human alpha-synuclein recombinant protein containing amino acids 1 -60 (Rpeptide, Bogart, Georgia) and human alpha- synuclein recombinant protein containing amino acids 1 -1 19.
• a lyophilized product from Rpeptide, Bogart, Georgia (Catalog number S-1001 -2) was used. This was dissolved in 20 mM tris and 300 mM NaCI buffer at concentration of 1 mg/ml protein.
• the protein solution was incubated 170 ⁇ aliquots in 96 well plate with a 70 ⁇ m diameter ceramic bead in each well at 200 rpm in Vortemp 56 shaker incubator (Labnet International, Edison, NJ, USA), at 37 °C for 7 days, and the formation of fibrils was followed by adding thioflavin T and measuring fluorescence in one of the wells.
• the recombinant alpha-synuclein containing amino acids 1 -60 was dissolved in water to give a concentration of 1 mg/ml.
• alpha-synuclein containing amino acids 1 -119 was made using the following construct: A synthetic gene coding for a 6 amino acid Histidine tag, followed by factor Xa cleavage site and sequence coding for human alpha-synuclein amino acids 1 -1 19:
• the expression vector was transformed into E. coli BL21 and a single colony picked for expression using the overnight express autoinduction system from Novagen (User protocol TB383 rev. H1005). The scale was 500 ml of final culture volume. Cells were harvested by centrifugation 10 min at 6000g and subsequently lyzed using BugBuster protein extraction Reagent (User protocol TB245 Rev. 0304). After lysis the sample was cleared by centrifugation and the supernatant used for further purification. [000168] The His-tagged protein was purified on a 5ml HisTrap column (GE healthcare) equilibrated in 20 mM Sodium phosphate pH7.5, 1 M NaCI (A-buffer).
• the purified his-tagged alpha-synuclein 1 - 1 19 was incubated with factor Xa in a 1 :50 ration using the Novagen kit (69037-3FRX). After overnight incubation, the factor Xa was removed batchwise using Xarrest agarose. The cleaved alpha-synuclein 1 -1 19 was finally purified by permissive HisTrap chromatography as described above. From the flow through the purified alpha- synuclein 1 -1 19 was obtained and concentrated to -400 ⁇ g /m ⁇ using centricon concentration devises.
• Alpha-synuclein (Rpeptide) was rehydrated in PBS at 2 mg/ml and peroxynitirite (100 ⁇ L /mg protein) was added dropwise while mixing. The nitrosylated alpha-synuclein was then dialyzed in 5L PBS and stored at -20 °C.
• Dopamine was used to oxidize alpha-synuclein. Equal volumes of a 200uM solution of Dopamine-HCL (Sigma P5244) prepared in 10mM PBS, pH7.4 and a 28 ⁇ solution of alpha-synuclein (Rpeptide) in 10mM PBS, pH7.4 were combined. The resulting 14 uM alpha-synuclein/100 uM Dopamine were incubated at 37 °C O/N (over night). The oxidized alpha-synuclein was then dialyzed in PBS and stored at - 20 °C. [000172] Different native and chimeric versions of synuclein proteins were produced in order to screen a diverse library of anti-alpha-synuclein antibodies.
• Screening constructs included the following: human, mouse, rat and cynomolgus monkey alpha-synuclein, human Beta-synuclein, Human Gamma-synuclein (fig 21 and 22) and lastly an alpha-synuclein derivative that lacked residues 120-140 of alpha- synuclein.
• a series of 4 shuffle constructs A-Syn-AAKK-BAP, A-Syn-BAAK- BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID Nos:11 -14) were produced.
• constructs contained linear stretches of human alpha-synuclein (A), human Beta-synuclein (B) and chicken alpha-synuclein (K).
• Gene were cloned containing a Biotin Acceptor Peptide (BAP) tag fused to the C-terminus of the ligands in order to facilitate site specific biotinylation of each of the ligands.
• BAP Biotin Acceptor Peptide
• Mammalian expression vectors were constructed carrying the different alpha-synuclein BAP tag fusion constructs (ASynBAP). The ligands were expressed in HEK 293 cells using transient transfection (Genmab A/S).
• Antibodies HuMab-Synuclein were derived from the immunizations of HuMAb mouse strain HCo17-BALB/c and HCo12-BALB/c mice, double knock out for the mouse immunoglobulin (Ig) heavy and mouse kappa light chain, which prevents the expression of antibodies that are completely murine (human monoclonal antibody; Medarex Inc., San Jose, CA, USA).
• the various mouse strains were made transgenic by the insertion of human Ig heavy and human Ig kappa light chain loci and differ in the number of human VH (variable domain of heavy chain) and VL (variable domain of light chain) genes.
• mice were immunized alternating intraperitoneally (IP) with 20 ⁇ g antigens and subcutaneously (SC, at the tailbase) with the same immunogen, with an interval of 14 days. A maximum of eight immunizations were performed, 4 IP and 4 SC.
• mice were additionally boosted twice intravenously (IV) with 10 ⁇ g alpha-synuclein immunogen protein in 100 ⁇ L PBS, four and three days before fusion.
• CFA complete Freund's adjuvant
• IFA incomplete Freund's adjuvant
• Antibody 37 came from an immunization protocol where human full length a-Synuclein-fibrils was used, alternating with alpha-synuclein C- terminally truncated forms with amino acids 1 -60 and 1 -1 19.
• Antibody 285 came from an immunization protocol where Human o Synuclein-monomer 1 -140 was used for the first 4 immunizations. If there was no titer, the immunization was continued with fibrils (ip/sc), otherwise it was continued with monomer.
• HuMAb mice with sufficient antigen-specific titer development as defined above were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and caval vein were collected. Fusion of splenocytes and lymph node cells with a mouse myeloma cell line was done by electrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions.
• Fused cells were seeded in fusion medium containing 10% Fetal Clone I Bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U/mL penicillin, 0.5 U/mL streptomycin (Cambrex), 50 ⁇ 2-mercaptoethanol (Invitrogen), 600 ng/mL interleukin 6 (IL-6) (Strathmann), 1 x HAT (Sigma) and 0.5 mg/mL kanamycin (Invitrogen) in HyQ mADCF-Mab (Perbio).
• the sera or supernatant potentially containing alpha-synuclein specific antibodies were added to the beads to allow binding to alpha-Synuclein and/or alpha-synuclein derived constructs.
• the binding of the anti-alpha-synuclein antibodies is detected using a fluorescent conjugate, Dyl_ight649 conjugated goat antihuman IgG, Fc specific.
• Two known mouse anti-alpha-synuclein antibodies, LB509 and Syn21 1 were included in screenings as positive controls.
• an anti-alpha-synuclein sera pool is used as a negative control in the 384 well format titer screening while human ChromPure IgG is used in the 1536 well format 8-bead based assay.
• Hybridoma cells from the best primary wells were seeded in semisolid medium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA). For each primary well, a well of a Genetix black 6-well plate was seeded. From each well, 25 sub clones were picked, using the ClonePix system (Genetix). The sub clones were picked in harvest medium. After seven days, the supernatants of the sub clones were screened again for
• Synuclein-specific human IgG binding and the human IgG concentration was measured using Octet (Fortebio, Menlo Park, USA). From each primary well, the best sub clone was selected and expanded in expansion medium containing only 600 ng/mL IL-6, 0.5 U/mL penicillin, 0.5 U/mL streptomycin and 1 x proHT. The sub clones were expanded from one 96-well plate well to one 24-well plate well to four 24-well plate wells to six 6-well plate wells. Clones derived by this process were designated as primary clones (PC).
• PC primary clones
• HuMabs Prior to coupling to amine reactive sensors, HuMabs were diluted in MES pH 6.0 buffer (18-5027). Coupling was performed at 30°C and 1000 rpm as follows: Amine reactive sensors were pre-wet in PBS and subsequently activated with EDC/NHS(ForteBio. Art.no. 18- 1033/18-1034) activation solution (according to manufacturer's instruction) for 300 seconds. Activated sensors were immobilized with HuMabs during 600 seconds.
• RNA was prepared from 0.2 to 5x106 hybridoma cells and 5'- RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA, using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturer's instructions.
• VH and VL coding regions were amplified by PCR and cloned directly, in frame, in the p33G1 f and p33Kappa expression vectors (containing the human lgG1 /kappa constant domain encoding sequences), by ligation independent cloning (Aslanidis, C. and P.J. de Jong, Nucleic Acids Res 1990;18(20): 6069-74).
• VL clones and 16 VH clones were sequenced. Clones with a correct Open Reading Frame (ORF) were selected for further study and expression. Vectors of all combinations of heavy chains and light chains were transiently co-expressed in FreestyleTM 293-F cells using 293fectin.
• ORF Open Reading Frame
• Comparator antibody 9E4 was generated based on the VH and VL sequence derived from hybridoma PTA-8221 (US patent 20080175838) (SEQ ID NO 42 and 43)
• Antibodies were produced by transfection in HEK293 6E cells using the pTT5 vectors and PEIpro as a transient transfection agent (National Research Council of Canada).
• the heavy and light chains were transfected into HEK293 cells using PEIpro (WVR), and cells were supplemented with TN1 (Sigma) 24 hours after transfection. Cells were grown until the viability approached 50%, and yield of antibody measured by easy IgG titre (Thermo). Culture supernatant was filtered over 0.2 ⁇ dead-end filters, loaded on 5 ml_ Protein A columns (rProtein A FF, Amersham
• ASynBAP was run at 100-3200nM and 25-3200RU, respectively. The highest concentration in each titration series was run in duplicate. The surface was regenerated with 10mM Glycine-HCI, pH 1 .7 (30sec inject) to remove captured mouse antibody and analyte in the end of each cycle. HBS-EP (GE Healthcare, Cat. No: BR-1001 -88) was used as running buffer and sample diluent in all experiments and the assay was run at 25 °C. All samples were kept at 4oC before acquisition. [000191] The response recorded in Fc1 , where capture antibody had been immobilized but no Alpha-Synuclein antibody captured, was subtracted from the response in Fc2. A 1 :1 or 2:1 binding algorithm was fit to the dataset using
• SBA antibody peroxidase conjugate
• ABTS 2,2'-azino-di-3-ethylbenzthiazoline sulfonate
• the color development was quantified with a charge coupled device (CCD) - camera and an image processing system.
• CCD charge coupled device
• image processing the values were obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader.
• the results were quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false- positive value, the cards are manually inspected and any values caused by an air- bubble are scored as 0.
• the binding data of antibody 37 and 285 to peptides containing the sequence ILEDMP or ILED respectively can be seen in Figure 7.
• Recombinant alpha-synuclein monomers and fibrils can be taken up by primary neurons in culture. As shown schematically in Figure 10, after uptake of the alpha-synuclein in neurons, it can be processed by intracellular proteases, such as Calpain I, with the major protease sensitive site at amino acid 1 19/122. To investigate truncation of alpha-synuclein by proteases mouse primary cortical neurons were prepared as described in Elvang et al. 2009 (Elvang et al.. J Neurochem.
• alpha-synuclein can be cleaved by Calpain-1 .
• the cleavage site of Calpain-1 for f ibrillized alpha-synuclein has been found to be in the region 1 14-122 (Mishizen-Eberz, J of Neurochem, 86, 836-847, 2003).
• the epitope of 285 overlaps with the epitope of 37 and it would also be expected to inhibit the protease cleavage.
• the amino acid sequence of 37v2 only differs from 37 at one amino acid in CDR and has similar binding as 37, so it would also be expected to inhibit protease cleavage in similar manner to 37.
• mouse primary cortical neurons prepared as above are plated in 96 well plates (15,000 cells per well).
• DIV in vitro culture
• 50% of media is changed and supplemented with cytosine arabinoside (final cone, of 1 uM).
• cytosine arabinoside final cone, of 1 uM
• half of the media is changed with glia conditioned media along with alpha synuclein fibrillary material, either crude fibril seeds or pure seeds.
• the crude fibril seeds are made from recombinant monomeric human alpha-synuclein, which was isolated from bacteria and the monomers were filtered through an Amicon Ultra 100.000 cut off filter (Millipore cat.
• Half of the media in the primary neurons is replaced with glia conditioned media every week to maintain them up to DIV21.
• the neurons are fixed and stained for Phospho-synuclein using a rabbit antibody specific for phosphorylation of alpha- synuclein at amino acid S129 (abeam 51253), followed by a fluorescently labelled anti- rabbit antibody, fluorescence is quantified using automated fluorescent microscopy, Cellomics Arrayscan. Nuclei were detected in one channel and defined the number of valid cells. Phosphorylated alpha-synuclein spots were detected in another channel in a pre-defined ring-formed area surrounding the nucleus, thus representing the cytoplasm of the cells. The average number of spots per cell was calculated.
• Fig 12A Example of cell staining is shown in Fig 12A. Phosphorylated alpha synuclein spots are not seen in untreated neurons. Neurons incubated with crude or pure seeds (1 -1 Ong per well) induce phosphorylation of alpha synuclein (Fig. 12A). In neurites, phosphorylated synuclein appears as spots or punctate and some of the phospho-synuclein in the neurites appear elongated.
• cells were harvested in phosphate buffered saline solution (PBS) and centrifuged. Pellet was resuspended in 1 % triton buffer with protease inhibitors. Samples were kept on ice for 15 min. followed by sonication. The samples were centrifuged at 100,000x g for 30 min. at 4C. The supernatant is collected and labelled as soluble fraction. The pellet was washed once in triton buffer and re- suspended in 1 % SDS buffer followed by sonication. Samples were centrifuged again at 100,000xg for 30 min. The supernatant is collected as insoluble fraction.
• PBS phosphate buffered saline solution
• FIG. 12 B shows the Western blot of the soluble and insoluble fraction from the primary neurons with and without crude seeds.
• Example 7 Acute Electrophysiological Effects Of Alpha-Synuclein Antibodies in vivo
• F28-snca transgenic and age-matched control male mice (CRO breeding, Taconic Europe A/S) aged 4 to 6 months were single-housed in controlled temperature (22 ⁇ 1 .5 oC) and humidity conditions (55-65%) and kept in a 12:12 hour light/dark cycle (lights on at 06:00h). Food and water were available ad libitum.
• mice were anesthetized with an intraperitoneal (i.p.) injection of urethane (1 .2 g/kg). Mice were then mounted in a stereotaxic frame, their temperature adjusted to 37.5 °C via a heating pad, and the skull was exposed.
• i.p. intraperitoneal
• a platinum wire was placed in the frontal bone to act as a reference, and an additional hole was drilled for insertion of the recording and stimulating electrodes in the hippocampus, at the following coordinates according to the atlas of Paxinos and Franklin (Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates, 4th Edition, 2001 ): recording, 1 .5-1 .7 mm posterior to Bregma, 1 .0-1 .2 mm lateral to the midline, 1 .4-1 .7 mm below the surface of the brain; stimulation, 1 .8-2.0 mm posterior to Bregma, 1 .5-1 .7 mm lateral to the midline, 1 .5-1 .7 mm below the surface of the brain. Animals were left in the stereotaxic frame through the whole duration of the recordings and their level of anesthesia was regularly checked.
• fEPSP Field potentials
• basal synaptic transmission was assessed by the relationship between stimulation intensity and slope of the evoked fEPSP (input-output relationship).
• the different intensities of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 ⁇ , and were applied successively in increasing order, with 2 to 3 repeats for each intensity.
• Basal synaptic transmission was found to be significantly impaired in F28-snca transgenic compared to age-matched control mice.
• Basal synaptic transmission in GM37- treated transgenic mice was not significantly different from basal synaptic transmission in PBS-treated littermates, indicating a full reversal of the impairment (Fig 15).
• GM285 also induced a significant reversal of the impairment in basal synaptic transmission in F28-snca transgenic mice (Fig 16). Basal synaptic transmission in GM285-treated transgenic mice was not significantly different from basal synaptic transmission in PBS-treated littermates, indicating a full reversal of the impairment.
• Example 8 Microdialysis to assess human alpha-synuclein in the brain of awake freely moving animals
• the push-pull microdialysis method was used to assess the levels of human alpha-synuclein in brain interstitial fluid (ISF). Mice were single-housed in controlled temperature (22 ⁇ 1 .5°C) and humidity conditions (55-65%) and kept on a 12:12 hour light/dark cycle (lights on at 06:00h). Food and water were available ad libitum. The current study was performed in the hippocampus of F28-snca transgenic mice (50-54 weeks old).
• ISF brain interstitial fluid
• mice were anaesthetized with isoflurane and an intracerebral guide cannula (CMA) was stereotaxically implanted into the brain, positioning the microdialysis probe in the hippocampus (co-ordinates of probe tip: 3.1 mm posterior and 2.8 mm lateral from bregma, and 1 .3 mm relative dura mater) according to the atlas of Paxinos and Franklin 2001 .
• Anchor screws and acrylic cement were used for the fixation of the guide cannulas. After implantation of the cannula mice were allowed to recover from the surgery for 2-3 days before dialysis.
• a 2-mm, 1000 kDa cut-off CMA probe was inserted through the guide cannula.
• a probe was connected to a microdialysis peristaltic pump with two channels (MAB20; Microbiotech) and operated in push-pull mode.
• the inlet tubing of the microdialysis probe was connected to a peristaltic pump perfusing the probe with artificial cerebrospinal fluid (aCSF; in mM: 147 NaCI, 2.7 KCI, 1 .2 CaCI 2 , 0.85 MgCI 2 ).
• the peristaltic pump was also connected to the outlet tubing in order to prevent perfusion fluid loss from the probe, by pulling the fluid through the tubing.
• GM37 ameliorate motor phenotype in rat Parkinson model.
• Targeted overexpression of human alpha-synuclein to dopaminergic neurons in the rat midbrain can be achieved using a recombinant adeno-associated viral vector (rAAV) and is associated with a progressive loss of dopaminergic cells in the substantia nigra as well as motor impairments.
• rAAV adeno-associated viral vector
• GM37 inhibits seeding of endogenous mouse alpha synuclein aggregation and phosphorylation
• mice 16;338(6109):949-53).
• GM37 alpha-synuclein specific monoclonal antibody GM37 could reduce the appearance of alpha-synuclein fibril-induced phosphorylated alpha synuclein inclusion formation in vivo a total of 45 mice were used. Mice were dosed with GM 37 at 30 mg/kg i.p, GM 3715 mg/kg i.v. , or vehicle ip (PBS). One day later the mice were anesthetized and stereotactically injected in one hemisphere with 2 ul of recombinant human alpha-Syn crude seeds, made as described previously (Example 6) (total of 2 ⁇ g crude seeds per animal).
• mice received weekly i.p. or i.v. injections of antibodies until sacrifice at 45 days. Groups of 15 mice/group were dosed either iv w. GM37 15mg/kg, ip with GM37 30mg/kg, or PBS (10 ml/kg) ip once weekly.
• the antibody levels in plasma are shown in Figure 20B and show a dose dependent increase in antibody plasma concentration and accumulation of antibody in plasma during the six weeks.
• the antibody levels in csf are shown in Figure 20C, and show that around 0.1 % of antibody level in plasma can be measured in csf.
• the mice were anesthetized, transcardially perfused with PBS, followed by perfusion with neutral buffered paraformaldehyde (4%). The brains were removed and incubated overnight for post-fixation in neutral buffered paraformaldehyde.
• Immunohistochemistry was performed on 45 ⁇ thick serial sections by Neuroscience associates. Briefly, Using MultiBrain® technology, up to 25 mouse brains were embedded together per block, into 3 blocks, freeze-sectioned at 45 ⁇ thickness in the coronal plane, and collected into cups containing antigen preserve solution. Every sixth section was stained with antibody to Ser-129 phosphorylated alpha-synuclein (Anti- alpha Synuclein (phospho S129) antibody [Psyn/81 A] ab184674) to reveal Ser129 phosphorylated alpha synuclein reactive structures.
• Anti- alpha Synuclein (phospho S129) antibody [Psyn/81 A] ab184674
• antibody GM37 could have therapeutic effect in PD by blocking entry of extracellular pathological alpha- synuclein into neurons, by blocking its propagation between neurons and/or facilitating clearance from the ISF by uptake into microglia.
• treatment with antibody GM37 could have a therapeutic effect on loss of dopaminergic cells and development of motor deficits in PD.
• Example 11 Manufactability of GM37 and GM37 variants
• the anti-alpha-synuclein antibodies are produced in mammalian cell culture under conditions that mimic the production conditions that will be used for producing clinical grade material for use in patients. It is well known that proteins produced in this manner undergo post-translational modifications that can impact both therapeutic potency of the antibody as well as biophysical attributes that affect the stability of the antibody over time. Empirical knowledge ascertained from decades of studies identified a set of post-translational modifications known to provide risk for the developability of a specific molecule. These post-translational modifications have been shown to correlate with amino acid strings present in the primary sequence of the heavy and light chain proteins.
• Algorithms have been generated that can identify these sequences and determine the potential risk they will have on the manufacturability and developability of a therapeutic antibody.
• In silico analysis of the primary sequence of the antibody can be used to de-risk a molecule for its potential to be developed as a therapeutic.
• detailed analysis of the VH and VL regions can identified unique amino acids that are deemed important for the molecules activity but also may be a potential risk for its stability over time.
• Sequence specific deamidation has been identified as a potential risk for protein structures. Protein deamidation can occur on the amide side chains of glutamines or asparagine residues and transform them into a carboxylate group (Lorenzo et al.
• Nonenzymatic deamidation at neutral pH occurs faster for asparagine and is therefore considered a higher risk than glutamine.
• the activity is further influenced by the subsequent amino acid in the sequence and can occur at a rate of days or years. The actual fate of the protein that undergoes deamidation needs to be evaluated experimentally to determine the impact of the change both on its stability and activity.
• Amino acid residues 54 is an asparagine(N) followed by a glycine(G) at position 55.
• the N54 is at high risk for spontaneous deamidation.
• transient transfections were performed using CHOK1 SV GS-KO cells which had been in culture for minimum 2 weeks. Cells were sub-cultured 24 hours prior to transfection. All transfections were carried out via electroporation using Gene Pulse XCell (Bio-Rad). For each transfection, viable cells were resuspended in pre-warmed CD-CHO media supplemented with 6mM L-glutamine to 2.86x10 7 cells/ml. 40 ⁇ g of each established SGV DNA containing the appropriate heavy and light chains were aliquoted into each cuvette (Bio-Rad, GenePulser cuvette, 0.4 cm gap, 165-2088) and 700 ⁇ cell suspension added.
• Cells were electroporated at 300V, 900 ⁇ . Transfected cells were transferred to rep-warmed media in Erienmeyer flasks and the contents of the cuvettes rinsed twice with prewarmed media were also transferred to the flasks. Transfectant cultures were incubated in a shaking incubator at 36.5 °C, 5% C0 2 , 85% humidity, 140 rpm for 6 days. Cell viability was measured at the time of harvest using a Cedex HiRes automated cell counter (Rosche).
• a competition assay was performed using preincubation of a fixed concentration (0.3 ⁇ g /m ⁇ ) of each of the following antibodies, GM37 (named GM 37wt), GM37 variantl , GM37 variant2 and GM37 variant3 with a range of 0-1000 nM human alpha-synuclein for 60 minutes at room temperature. The remaining unbound antibody was captured and measured on ELISA plates coated with 100 ng/ml of recombinant human alpha-synuclein using an anti-human detection antibody by
• the IC50s of the interaction are 1 .9nM, 1 .6nM, 2.1 nM and 1 .4nM for GM37 wt, GM37variant1 , GM37variant2 and GM37variant3, respectively (as determined using Prism Graphpad®).

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  • 2017-11-14 EP EP17807745.9A patent/EP3541836A1/de active Pending
  • 2017-11-14 AU AU2017359944A patent/AU2017359944A1/en active Pending
• 2019
  • 2019-05-09 CO CONC2019/0004755A patent/CO2019004755A2/es unknown
  • 2019-05-10 CL CL2019001293A patent/CL2019001293A1/es unknown
  • 2019-05-13 IL IL266596A patent/IL266596A/en unknown
• 2020
  • 2020-12-01 US US17/109,028 patent/US20210236634A1/en active Pending
• 2021
  • 2021-12-10 CL CL2021003306A patent/CL2021003306A1/es unknown

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